Viruses Affect Cell Reprogramming

Viral vectors used to carry transcription factors that de-differentiate cells into a stem-cell-like state are themselves a key factor in efficient reprogramming.

By Sabrina Richards | October 25, 2012

Wikimedia, Nissim Benvenisty.Triggering a pathway designed to sense viral infection can help boost generation of induced pluripotent stem cells (iPSCs), suggesting that the viruses used by many reprogramming methods influence the fate of the cell. The new research, published today (October 25) in Cell, suggests that targeting the pathway without using viruses could avoid the risk that the viruses’ genetic material will integrate into the genome and cause the cell to become cancerous—a common concern for iPSC therapies.

“This is something everyone else missed before,” said Gioacchino Natoli, an experimental oncologist at the European Institute of Oncology in Italy who was not involved in the study. An innate immune response to the viral vector is clearly needed for reprogramming, and “stimulating this leads to reprogrammed cells without exogenous DNA being integrated,” he added.

Six years ago, Shinya Yamanaka identified four key transcription factors, that, when transduced into cells using a viral vector, caused cells to de-differentiate into a stem-cell-like state, capable of generating a multiplicity of human tissues. He shared this year’s Nobel Prize in Medicine for the achievement, which has already made significant contributions to biomedical research. But there was a major hurdle on the path to clinical use: the strategy relies on a retrovirus that can integrate into host genomes, sometimes activating an oncogene that could render the cell cancerous. Injecting such cells as therapies thus raised concerns about the risk of patients developing tumors.

Hoping to devise a non-integrative carrier for Yamanka’s reprogramming factors, John Cooke, who studies vascular regeneration at Stanford University and colleagues created cell-permeant peptides (CPPs)—the transcription factors attached to an 11-amino–acid-long protein tag that allows them to pass through the cell membrane. But using CPPs to reprogram fibroblasts turned out to be a hundred times less efficient than using retroviral vectors. Compared to virally-transduced cells, CPP-programmed cells also expressed critical pluripotency target genes several days later and at much lower levels.

It occurred to Cooke’s postdocs Jieun Lee and Nazish Sayed that the viral vectors themselves might be sending an important signal to the fibroblasts the researchers were trying to reprogram. So they added a GFP-expressing viral particle during their CPP reprogramming protocol—and found that the virus boosted efficiency, even though the CPPs carried the transcription factors.

Sayed and Lee hypothesized that the viral particles were actually stimulating innate immune pathways in the fibroblasts. A key player in the activation of these pathways is the toll-like receptor 3 (TLR3), which is known to be activated by the double-stranded viral RNA (dsRNA) carried by the viruses. Sure enough, knocking down TLR3 reduced the efficiency of a retroviral reprogramming. Furthermore, adding a synthetic mimic of dsRNA during CPP reprogramming had the reverse effect, boosting the number of human fibroblasts that completed the transition to iPSCs.

The researchers were even able to nail down the mechanism. It turns out that TLR3 stimulation leads to epigenetic changes that allow easier transcription factor binding to their target genes.

“In fibroblasts, much of the genome is in a closed conformation, so transcription factors have trouble accessing promoters,” explained Cooke. “But activation of innate immunity [by TLR3] puts chromatin in an open configuration so transcription proteins work.” It may also be possible to target the chromatin directly, he noted.

It’s not the first time epigenetic marks have been fingered as key players in cellular reprogramming, and it’s probably not the last. “It will be interesting to use genome-wide epigenetic studies to elucidate the epigenetic similarity between embryonic stem cells (the gold standard of pluripotency) and reprogrammed cells in the presence and absence of active TLR3 stimulation,” Kitai Kim, a cancer and stem cell biologist at Memorial Sloan-Kettering Cancer Center, wrote in an email.

Additionally, the work may have implications for the origin of cancer. “iPSC reprogramming and tumorigenicity share noticeable levels of the similarity,” said Kim, who did not participate in the research. Cooke and his colleagues agree and are planning to study how inflammation may help create an open chromatin state that enables new differentiation pathways. “The state of chronic inflammation is often associated with cancer,” Cooke noted, speculating that inflammation may push chromatin toward a plastic state that enables malignant transformation.

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My perception (ALWAYS open to being updated and upgraded by new empirical findings) is that the entire field of human evolution is only just now beginning to rocket forward.

This article adds still one more new incremental curve along the road to understanding of mechanisms that modify, or depart from, what have been theoretical arguments shaped by unfalsifiable convictions.

Let empirical science show what it will show, and not be hampered by any prior convictions about what may be implications of former states of inadequate data.

Our data in the sciences will never exhaust each and every iota of reality.

To study differential equations in mathematics, and to extend from them how the progress of a formula can proceed in one direction for a time and then veer off in another direction, or suddenly wax into a line that goes off into chaotic or irrational results, demonstrates (I believe) how we can be wrong in hoping to predict the future course of yet untested (or yet unfalsifiable) assumptions.

We are not wrong to make such extensions, and test them. Where we have derived only such data as we have on hand at any given junctur in the progress of mankind's search for understanding nature (science), we do well to extend from what we have, in arriving at new questions, at dreaming up new hypotheses, in testing what extensions of our data-so-far suggest may lie ahead in our learning. But if we allow those extensions from evidence we have on hand so far to become dogmatic, we stultify thereby the promise that consists in open mind.

Let us find, and follow, new data cybernetically, and not insist that it will lead where we predict.

Let us each choose for ourselves what we wish to believe and test in our thinking as working hypotheses, only, and not as dogma.

Whatever is ahead, in the quest for human knowledge, let it reveal what it will reveal, and not be cramped by being forced to fit the alternative assumptions so far unfalsifiable.

The extent to which biological evolution remains to be discovered will be mined accurately not by our predictions or convictions but, rather, by our future results.

Re: "It’s not the first time epigenetic marks have been fingered as key players in cellular reprogramming, and it’s probably not the last."

In two science fiction novels: "Darwin's Children" (2003) and "Darwin's Radio" (1999), Greg Bear incorporated the concept of viral-induced epigenetic changes in cellular reprogramming into stories about the evolution of a new species of human.

It has since become clearer that the epigenetic effects of virus-driven changes in intracellular signaling and stochastic gene expression (e.g., due to microRNA/mRNA - driven intermolecular changes) are associated with nutrient-chemical dependent cell survival and pheromone-controlled reproduction in species from microbes to man.

If, as suggested here, virus-driven inflammation pushes chromatin toward a plastic state that enables malignant transformation, the control of cellular and organism-wide homeostasis by the epigenetic effects that inhibit viral replication (e.g., proper nutrition and social stress inhibition), might be the best approach to prevent our evolution to another species, or our diet-driven and social stress-driven extinction.

Genes are instructions as in recipes. The regulation of those genes is what is now being called epigenetics, usually with the proviso that the regulatory changes can persist after the inducing conditions have gone. Regulation is not evolution, it is one of the possible programmable responses to current conditions, and only alters the extent to which a gene is used, it doesn't change the underlying instruction. It may persist for minutes or a lifetime or even a generation or two, but is still only a switch and can be reversed just as easily. Evolution requires a mutation (change in the base sequence) of DNA and then loss of the original sequence from the population. Evolution is not exactly reversible, an epigenetic switch is exactly reversible. If you set your word processor to produce blue text instead of black, it will continue to produce blue text until you set the text colour switch back. You have not evolved a new word processor by setting it to produce blue text if you never tried to reset it, you couldn't tell whether it was still resettable or not, I suppose.

Your post will be rather good and I'm sure some will find it interesting because it's about a topic that's as widely discussed as others with insurance. Some may even find it useful.Thanks so much for your post.

Re: "Evolution requires a mutation (change in the base sequence) of DNA and then loss of the original sequence from the population."

What you described is ecological adaptation that arises from the conserved molecular mechanisms of the biophysically constrained chemistry of nutrient-dependent protein folding. Protein folding is perturbed by mutations, which is why they do not lead to increasing organismal complexity. They lead to physiopathology that alters the pheromone-controlled physiology of nutrient-dependent reproduction in species from microbes to man, which is how most mutations are removed.

Biologically-based cause and effect has now been linked from viral microRNAs to cel type differentiation in all cells of all individuals of all species. See: TSLive: The Enemy Within